Gram-positive alkaliphilic microorganisms

ABSTRACT

The present invention provides novel aerobic, Gram-positive alkaliphilic bacteria which have been isolated from in and around alkaline soda lakes. These alkaliphiles have been analyzed according to the principles of numerical taxonomy with respect to each other and also to a collection of known bacteria. In addition, these bacterial taxa are further circumscribed by an analysis of the lipid components which serve as chemotaxonomic markers. The alkaliphiles of the present invention produce alkalitolerant enzymes which are capable of performing their functions at high pH which makes them uniquely suited for applications requiring such extreme conditions.

This application is a divisional of U.S. Ser. No. 08/914,736 filed Aug.19, 1997 now U.S. Pat. No. 5,858,748 which is itself a divisional ofU.S. Ser. No. 08/314,045 filed Sep. 28, 1994 now U.S. Pat. No. 5,707,851which is a divisional of U.S. Ser. No. 07/903,786 filed Jun. 24, 1992now U.S. Pat. No. 5,401,657 which is a continuation-in-part of U.S. Ser.No. 07/719,307 filed Jun. 24, 1991, now abandoned which is acontinuation-in-part of U.S. Ser. No. 07/562,863 filed Aug. 6, 1990, nowabandoned. The contents of these applications are incorporated herein byreference.

The present invention is in the field of microbiology and moreparticularly in the field of alkaliphilic microorganisms.

BACKGROUND OF THE INVENTION

Alkaliphiles are defined as organisms which exhibit optimum growth in analkaline pH environment, particularly in excess of pH 8, and generallyin the range between pH 9 and 10. Alkaliphiles may also be found livingin environments having a pH as high as 12. Obligate alkaliphiles areincapable of growth at neutral pH.

Alkaliphiles may be found in certain everyday environments such asgarden soil, presumably due to transient alkaline conditions caused bybiological activities including ammonification, sulphate reduction orphotosynthesis. A much richer source of a greater variety ofalkaliphilic organisms may be found in naturally occurring, stablealkaline environments such as soda lakes.

A more detailed study of soda lakes and alkaliphilic organisms ingeneral is provided in Grant, W. D., Mwatha, W. E. and Jones, B. E.((1990) FEMS Microbiology Reviews, 75, 255-270), the test of which ishereby incorporated by reference. Lists of alkaline soda lakes may befound in the publications of Grant, W. D. and Tindall, B. J. in Microbesin Extreme Environments, (eds. R. A. Herbert and G. A. Codd); AcademicPress. London, (1986), pp. 22-54); and Tindall, B. J. in HalophilicBacteria, Volume 1, (ed. F. Rodriquez-Valera); CRC Press Inc., BocaRaton, Fla., (1988), pp. 31-70, both tests are also hereby incorporatedby reference.

Alkaliphiles, the majority of which are Bacillus species, have beenisolated from non-saline environments and are discussed by Horikoshi, K.and Akiba, T. in Alkalophilic Microorganisms (Springer-Verlag, Beriln,Heidelberg, N.Y., (1982). However, alkaliphilic organisms from salineand alkaline environments such as lakes are not discussed therein.Strictly anaerobic bacteria from alkaline, hypersaline, environmentshave been recently described by Shiba, H. in Superbugs (eds. K.Horikoshi and W. D. Grant); Japan Scientific Societies Press, Tokyo andSpringer-Verlag, Berlin, Heidelberg, N.Y., (1991), pp. 191-211; and byNakatsugawa. N., ibid, pp. 212-220.

Soda lakes, which may be found in various locations around the word, arecaused by a combination of geological, geographical and climaticconditions. They are characterized by the presence of large amounts ofsodium carbonate (or complexes thereof) formed by evaporativeconcentration, as well as by the corresponding lack of Ca²⁺ and Mg²⁺which would remove carbonate ions as insoluble salts. Other salts suchas NaCl may also concentrate resulting in environments which are bothalkaline and saline.

Despite this apparently harsh environment, soda lakes are neverthelesshome to a large population of prokaryotes, a few types of which maydominate as permanent of seasonal blooms. The organisms range fromalkaliphilic cyanobacteria to haloalkaliphilic archaeabacteria.Moreover, it is not unusual to find common types of alkaliphilicorganisms inhabiting soda lakes in various widely dispersed locationsthroughout the world such as in the East African Rift Valley, in thewestern U.S., Tibet, China and Hungary. For example, natronobacteriahave been isolated and identified in soda lakes located in China (Wang,D. and Tang, Q., “Natronobacterium from Soda Lakes of China” in RecentAdvances in Microbial Ecology (Proceedings of the 5th InternationalSymposium on Microbial Ecology, eds. T. Hattori et al.); JapanScientific Societies Press, Tokyo, (1989), pp. 68-72) and in the westernU.S. (Morth, S. and Tindall, B. J. (1985) System. Appl. Microbiol., 6,pp. 247-250). Natronobacteria have also been found in soda lakes locatedin Tibet (W. D. Grant, unpublished observations) and India (Upasant, V.and Desai, S. (1990) Arch. Microbiol., 154, pp. 589-599).

Alkaliphiles have already made an impact in the application ofbiotechnology for the manufacture of consumer products. Alkalitolerantenzymes produced by alkaliphilic microorganisms have already found usein industrial processes and have considerable economic potential. Forexample, these enzymes are currently used in detergent compositions andin leather tanning, and are foreseen to find applications in the food,waste treatment and textile industries. Additionally, alkaliphiles andtheir enzymes are potentially useful for biotransformations, especiallyin the synthesis of pure enantiomers. Also, many of the microorganismsdescribed herein are brightly pigmented and are potentially useful forthe production of natural colorants.

SUMMARY OF THE INVENTION

The present invention concerns novel aerobic, Gram-positive alkaliphilicbacteria. These bacteria have been isolated from samples of soil, water,segment and a number of other sources, all of which were obtained fromin and around alkaline soda lakes. These alkaliphiles have been analyzedaccording to the principles of numerical taxonomy with respect to eachother and also to a collection of known bacteria in order to confirmtheir novelty. In addition, these bacterial taxa are furthercircumscribed by an analysis of the lipid components which serve aschemotaxonomic markers.

The present invention also provides data as to the composition of theenvironments from which the samples containing the microorganisms wereobtained, as well as the media required for their efficient isolationand culture such that one of ordinary skill may easily locate such anenvironment and be able to isolate the organisms of the presentinvention by following the procedures described herein.

It is also an object of the present invention to provide microorganismswhich produce alkalitolerant enzymes. These enzymes are capable ofperforming their functions at high pH which makes them uniquely suitedfor applications requiring such extreme conditions. For example,alkalitolerant enzymes may be employed in detergent compositions, inleather tanning and in the food, waste treatment and textile industries,as well as for biotransformations such as the production of pureenantiomers and the production of natural pigments.

The genes encoding these alkalitolerant enzymes may be isolated, clonedand brought to expression in compatible expression hosts to provide asource of larger yields of enzyme products which maybe, if desired, moreeasily purified and used in various industrial applications, should thewild-type strain fail to produce sufficient amounts of the desiredenzyme, or performs poorly under norma, industrial fermentationconditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Simplified dendrogram showing clusters (phenons) obtained withthe S_(G) coefficient and Unweighted Average Linkage procedure.

FIG. 2. Simplified dendrogram showing clusters (phenons) obtained withthe S_(J) coefficient and Unweighted Average Linkage procedure.

FIG. 3. Simplified dendrogram showing clusters (phenons) obtained withthe S_(SM) coefficient and Unweighted Average Linkage procedure.

FIG. 4. Simplified dendrogram obtained with the S_(SM) coefficient andUnweighted Average Linkage procedure suing the derived minimumdiscriminatory tests.

DETAILED DESCRIPTION OF THE INVENTION Sampling

Several hundreds of strains of bacteria have been isolated from samplesof soil, water, sediment and a number of other sources in and aroundalkaline lakes. These samples were obtained as part of an investigationover a period of three years. The isolated bacteria are non-phototrophiceubacteria. Until now, such bacteria have not been well characterized.

The samples were collected in sterile plastic bags, Sampling wasconducted at lakes Elmenteita, Nakuru, Bogoria, Crater (Sonachi), LittleNaivasha (Oloidien) and Magadi, all of which are located in Kenya, EastAfrica, Alkaline soda lakes having similar environments may also befound in Tibet, China, Hungary and the western U.S. At each samplingsite, the physical appearance of the site and the sample were describedand physical parameters such as pH, conductivity and temperature weremeasured. Some of the samples were treated locally within 36 hours ofcollection of the samples but the majority were examined off-site,several weeks after collection.

Table 1 lists various strains which have been isolated. The strains arelisted according to the location from which the sample was taken and thephysical appearance of the sample itself.

Table 2 provides examples of typical chemical analyses of the lakewaters at the sampling locations at the time of extraction of many ofthe samples. These data are consistent with earlier analyses (Grant, W.D. and Tindall, B. J., supra).

Table 3 provides a list of the isolated strains arranged according tothe results of the numerical taxonomic analysis (FIG. 1). Furthermore,Table 3 provides physical properties of the original sample, inparticular the temperature, conductivity and alkaline pH, as well as thenumerous isolation media required for obtaining pure cultures of thesenovel bacteria. These media are letter coded with reference to AppendixA.

Tables 1, 2 and 3 provide data from which the environment of thesampling locations may be characterized. The chemical and physicalanalyses of the samples confirm the presence of alkaline pH, as well asthe presence of unusually high levels of Na₂CO₃, coupled with low levelsof Ca²⁺ and Mg²⁺. It is known that the basic environments of soda lakesare stable with respect to their pH and ionic composition. Moreover, themicrobial populations found at these sites remain largely stable, Thus,it is to be expected that the environment from which bacteria accordingto the present invention may be obtained can be determined from the datapresented in Tables 1-3.

The fresh soda-lake water samples were plated out on an alkalinenutrient medium (Medium A) soon after collection. Microscopic inspectionof the soda lake samples showed a surprisingly high diversity ofbacterial types. Considering the extremely alkaline nature of theenvironment, viable counts showed unexpectedly high numbers oforganotrophic bacteria, in the range of 10⁵-10⁴ colony forming units perml. The samples were stored either cooled or at ambient temperatures.After a few weeks' storage, the total numbers of bacteria in the samplerose, whereas the diversity of types decreased.

TABLE 1 Alkaliphilic Strains Arranged According to Their Place of OriginSTRAINS SAMPLE LOCATION SAMPLE APPEARANCE 3E.1 Lake Elmenteita Mud fromdried up (east bay). lake bed. wE1, wE2, Lake Elmenteita Sediment andwater, wE4 (east bay). littoral zone. 60E.4 Lake Elmenteita Mud,littoral (east bay). zone. wN10, wN12, Lake Nakuru, Mud and water, wN16north beach littoral zone. between Hippo Point and Njoro Point. wB3 LakeBogoria, Mud and water, northern mud littoral zone. flats. 66B.4 LakeBogoria Soda crusts and (west shore), mud (around hot Loboru deltaspring). area. 69B.4 Lake Bogoria Water column and (south bay).sediment, littoral zone. 13C.1, Crater Lake Mud and water, 71C.4, 72C.4(north point). littoral zone. 14LN.1, Little Lake Water column 15LN.1,Naivasha and sediment 79LN.4 (south shore). 80LN.4, 81LN.4 Little LakeBlack mud, benthic Naivasha zone. (south shore). 23M.1 Lake Magadi Mudand water. (causeway, upper western arm).

TABLE 2 Typical Chemical Analysis of Kenyan Lake Waters⁺ Na⁺ K⁺ Ca²⁺Mg²⁺ SiO₂ Cl⁻ SO₄ ²⁻ CO₃ ²⁻ TON* TA§ Lake (mM) (mM) (mM) (mM) (mM) PO₄³⁻ (mM) (mM) (mM) (mM) (mM) Elmenteita 196 3.58 0.07 b.l.d. 2.91 0.0365.1 2.0 68.0 0.8 119 Nakuru 326 5.63 0.15 b.l.d. 3.25 0.15 57.5 0.5198.3 1.9 259 Bogoria 796 6.78 0.19 0.01 1.98 0.17 115.5 1.1 516.7 0.5669 Crater 140 8.95 0.06 0.01 2.13 0.04 12.4 0.8 90.0 1.1 133 Little 8.71.79 0.28 0.65 1.02 0.003 4.8 0.5 <10.0 <0.07  18 Naivasha Magadi 282626.1 0.03 0.01 7.1 0.23 1124 12.8 1816 5.4 180 b.l.d. = below the limitsof detection * = Total Organic Nitrogen § = Total Alkalinity inmilliequivalents/liter ⁺ = October 1988

TABLE 3 Origin of the Strains Arranged by Cluster SAMPLE ISOLA- Conduct-TION CLUS- LOCA- Temp. ivity MED- TER STRAIN TION pH 0° mS/cm IUM 13E.1^(CT) Elmenteita  9.5 35  2 A 1 71C.4 Crater 10 26  10.2 E 1 81LN.4Little  8.5-9 30  1.2 D Naivasha 1 60E.4 Elmenteita 10 32  12.7 B 1 wE4Elmenteita n.t. n.t. n.t. A 2 69B.4 Bogoria 10.5 33  44 C 2 RS11^(CT) *2 RS14 * 2 Exig. * 2 RS13 * 3 wE1 Elmenteita n.t. n.t. n.t. A 3 wN10Nakuru n.t. n.t. n.t. A 3 wN12 Nakuru n.t. n.t. n.t. A 3 wN16^(CT)Nakuru n.t. n.t. n.t. A 4 13C.1 Crater  9.0 30  10 A 4 23M.1 Magadi 1136 100 A 4 14LN.1 Little  8.5 26  1 A Naivasha 4 15LN.1^(CT) Little  8.526  1 A Naivasha — wE2 Elmenteita n.t. n.t. n.t. A — wB3 Bogoria n.t.n.t. n.t. A — BG114 * 5 66B.4 Bogoria n.t. n.t. n.t. F 5 AB30 * 5RS10^(CT) * 5 RS17 * 5 AB49 * 5 AB42 * 6 RS7 * 6 RS8 * 6 RS15 * 6 RS16 *— 79LN.4 Little  8.5-9 30  1.2 F Naivasha — RS12 * — 72C.4 Crater 10 26 10.2 E — 80LN.4 Little  8.5-9 30  1.2 G Naivasha — Br.li * — Micro *n.t. = not tested The letter codes given for the Isolation Media referto Appendix A. The asterisk (*) refers to a reference strain; theidentity of which is provided in Table 4 (below).

Treatment of the Samples; Enrichment and Isolation of AlkaliphilicBacteria

A wide diversity of enrichment and isolation methods were applied. Someof the methods were specifically designed for the enrichment andisolation of alkaliphilic bacteria which exhibit specific types ofenzyme activity at an alkaline pH. Other techniques of a more generalnature were applied for the isolation of diverse sorts of alkaliphilicbacteria. In some cases, the specific conditions prevailing in the lakes(Table 2) were taken into account when experiments were performed forthe isolation of bacteria.

The different nutrient media employed for the isolation of the newalkaliphilic bacteria are designated Medium A—Medium G. The compositionof the various media employed is shown in Appendix A.

For the isolation of non-specific alkaliphilic organotrophic bacteria,soda-lake water samples or dilutions thereof were streaked out on analkaline nutrient agar, pH 10-pH 10.5 (Medium A). Samples of a moresolid consistency, mud, sediment, etc. were first suspended in analkaline nutrient broth (Medium A) before spreading on an alkalinenutrient agar (Medium A). The bacteria were cultivated in a heatedincubator, preferably at 37° C. In some cases, the samples weresuspended in an alkaline nutrient broth (Medium A) and the bacteriacultivated by shaking, preferably at 37° C. for 2-3 days beforespreading the broth onto an alkaline nutrient agar (Medium A) for theisolation of bacterial colonies.

For the isolation of alkaliphilic bacteria exhibiting specific types ofenzyme activity, samples were spread onto alkaline nutrient agarcontaining specific substrates such as lactalbumin or casein or oliveoil. In some instances, the bacteria in the sample were enriched for 1day up to several weeks in a non-specific alkaline nutrient broth suchas Medium A before spreading the broth onto an alkaline nutrient agarspecific for the detection of bacteria exhibiting enzyme activities suchas lipolytic or proteclytic activity.

Taxonomic Analysis

Twenty strains of bacteria isolated from in and around alkaline lakeswere assigned to the type of bacteria known as Gram-positive bacteria onthe basis of (1) the Dussault modification of the Gram's stainingreaction (Dussault, H. P., (1955), Journal of Bacteriology, 70,484-485); (2) the KOH sensitivity test (Gregersen, T., (1978), EuropeanJournal of Applied Microbiology and Biotechnology 5, 123-127; Halebian,S. et al., (1981), Journal of Clinical Microbiology, 13, 444-448); (3)the aminopeptidase reaction (Cerny, G., (1976), European Journal ofApplied Microbiology, 3, 223-225; ibid, (1978), 5, 113-122); and in mostcases, confirmation also on the basis of (4) a quinone analysis(Collins, M. D. and Jones, D., (1981), Microbiological Reviews, 45,316-354) using the method described by Collins, M. D. in ChemicalMethods in Bacterial Systematics (eds. Goodfellow, M. and Minnikin, D.)pp. 267-288, Academic Press, London, 1985.

The twenty strains were tested for 200 characters. The results wereanalyzed using the principles of numerical taxonomy (Sneath, P. H. A.and Sokal, R. R., in Numerical Taxonomy, W. H. Freeman & Co., SanFrancisco, 1973). The characters tested and manner of testing arecompiled in Appendix B. In addition, Appendix C records how eachcharacter was coded for taxonomic analysis.

As controls, 17 known Gram-positive bacteria were subjected to the sameanalysis using the same conditions where appropriate. These referencebacteria included genera that are known to include facultative orobligate alkaliphilic species. These 17 known reference bacteria arerecorded in Table 4 from which it will be seen that the “Type Strain” ofthe known species has been used where available. Thirteen of the strainsare known alkaliphilic Bacillus species.

TABLE 4 Gram-Positive Reference Strains * (RS7) (alkaliphilic) Bacillusspecies DSM 2514 (RS8) (alkaliphilic) Bacillus species DSM 2515 (RS10)(alkaliphilic) Bacillus species DSM 2517 (RS11) (alkaliphilic) Bacillusspecies DSM 2518 (RS12) (alkaliphilic) Bacillus species DSM 2519 (RS13)(alkaliphilic) Bacillus species DSM 2521 (RS14) (alkaliphilic) Bacillusspecies DSM 2523 (RS15) (alkaliphilic) Bacillus species DSM 2525 (RS16)Bacillus alcalophilus ^(†) DSM 485 (RS17) Bacillus alcalophilus subsp.halodurans DSM 497 (AB30) (alkaliphilic) Bacillus species ATCC 21596(AB42) (alkaliphilic) Bacillus species ATCC 21833 (AB49) (alkaliphilic)Bacillus species ATCC 21591 (Exig) Exiguobacterium aurantiacum ^(†)NCIMB 11798 (BG114) Arthrobacter luteus ATCC 21596 (Br.li)Brevibacterium linens ^(†) NCIMB 9904 (Micro) Micrococcus luteus ^(†)NCTC 2665 *abbreviation used in Figure 1, Figure 2 and Figure 3^(†)denotes “Type Strain”

Analysis of Test Data

The Estimation of Taxonomic Resemblance

The phenetic data, consisting of 200 unit characters was scored asindicated in Appendix C, and set out in the form of an “h x t” matrix,whose t columns represent the t bacterial strains to be grouped on thebasis of resemblances, and whose n rows are the unit characters.Taxonomic resemblance of the bacterial strains was estimated by means ofa similarity coefficient (Sneath, P. H. A. and Sokal, R. R., NumericalTaxonomy, supra, pp. 114-187). Although many different coefficients havebeen used for biological classification, only a few have found regularuse in bacteriology. We have chosen to apply three associationcoefficients (Sneath, P. H. A. and Sokal, R. R., ibid, p. 129 et seq.),namely, the Gower, Jaccard and Simple Matching coefficients. These havebeen frequently applied to the analysis of bacteriological data and havea wide acceptance by those skilled in the art since they have been shownto result in robust classifications.

The coded data were analyzed using the TAXPAK program package (Sackin,M. J., “Programmes for classification and identification”, In Method inMicrobiology, Volume 19 (eds. R. R. Colwell and R. Grigorova), pp.459-494, Academic Press, London, (1987)) run on a DEC VAX computer atthe University of Leicester, U.K.

A similarly matrix was constructed for all pairs of strains using theGower Coefficient (S_(G)) with the option of permitting negative matches(Sneath, P. H. A. and Sokal, R. R., supra, pp. 135-136) using theRTBNSIM program in TAXPAK. As the primary instrument of analysis and theone upon which most of the arguments presented herein are based, theGower Coefficient was chosen over other coefficients for generatingsimilarity matrices because it is applicable to all types of charactersor data, namely, two-state, multistate (ordered and qualitative), andquantitative.

Cluster analysis of the similarity matrix was accomplished using theUnweighted Pair Group Method with Arithmetic Averages (UPGMA) algorithm,also known as the Unweighted Average Linkage procedure, by running theSMATCLST sub-routine in TAXPAK.

The result of the cluster analysis is a dendrogram, a simplified versionof which is provided in FIG. 1. The dendrogram illustrates the levels ofsimilarity between the bacterial strains. The dendrogram is obtained byusing the DENDGR program in TAXPAK.

The phenetic data, omitting multistate characters (characters 1-5, 12,13; Appendix C) and thus consisting of 193 unit characters, and scoredin binary notation (positive=1, negative=0) where re-analyzed using theJaccard Coefficient (S_(j)) (Sneath, P. H. A. and Sokal, R. R, ibid, p.131) and Simple Matching Coefficient (S_(SM)) (Sneath, P. H. A. andSokal, R. R., ibid, p. 132) by running the TRBSIM program in TAXPAK. Afurther two dandrograms were obtained by using the SMATCLST with UPGMAoption and DENDGR sub-routines in TAXPAK. Simplified versions of thesedendrograms are illustrated in FIG. 2 and FIG. 3 respectively.

Results of the Cluster Analysis

S_(C)/UPGMA Method

FIG. 1 illustrates the results of the cluster analysis, based on theGower Coefficient and the UPGMA method, of 20 Gram-positive,alkaliphilic bacteria isolated from in and around alkaline lakes,together with 17 known Gram-positive bacteria, including 14 alkaliphilicspecies.

Six natural clusters or phenons of alkaliphilic bacteria are generatedat the 79% similarity level. These six clusters include 15 of the 20alkaliphilic bacteria isolated from alkaline lakes. Although the choiceof 79% for the level of delineation may seem arbitrary, it is in keepingwith current practices in numerical taxonomy (Austin, B. and Priest, F.,in Modern Bacterial Taxonomy, p. 37; Van Nostrand Reinhold; Wokingham,U. K., (1986)). Placing the delineation at a lower percentage wouldcombine groups of clearly unrelated organisms whose definition is notsupported by the data. At the 79% level, 3 of the clusters exclusivelycontain novel alkaliphilic bacteria representing 13 of the newlyisolated strains, and these may represent new taxa.

As excepted, the cluster analysis groups the control Bacillus species in3 distinct clusters which are separate from the novel alkaliphilicbacteria of the present invention. These results broadly concur with ataxonomic analysis of alkaliphilic Bacillus strains reported by Fritze,D., et al. (International Journal of Systematic Bacteriology, (1990),40, 92-97)). None of the known organisms are significantly related toany of the 3 clusters of new, Gram-positive alkaliphilic bacteria. Aclear discrimination between these clusters is possible using theconcept of the minimum discriminatory tests (see below) andchemotaxonomic information (see below).

The significance of the clustering at this level is supported by theresults of the TESTDEN program. This program tests the significance ofall dichotomous pairs of clusters (comprising 4 or more strains) in aUPGMA generated dendrogram with squared Euclidean distances, or theircomplement as a measurement and assuming that the clusters arehyperspherical. The critical overlap was set at 0.25%, As can be seenfrom Table 5, the separation of the clusters is highly significant.

TABLE 5 Significance of the Clusters Generated by the S_(G)/UPGMA MethodProvided by TESTDEN CLUSTER separates from CLUSTER at Significance level1 2 P = 0.99 2 3 0.99 <P> 0.95 3 4 + wE2 P = 0.99 5 6 P = 0.99

The cophenetic correlation is 0.804 which indicates the high degree ofreliability with which this dendrogram represents the true taxonomicstructure. (Sneath, P. H. A. and Sokal, R. R., supra, pp. 277-280, 304).Furthermore, the pattern of clusters obtained using the JaccardCoefficient (FIG. 2 and below) and Simple Matching Coefficient (FIG. 3and below) support the conclusions drawn here.

Two of the newly isolated alkaliphiles, 69B.4 and 66B.4 cluster amongthe alkaliphilic Bacillus species and may properly be considered tobelong to the genus Bacillus. However, five of the new alkaliphilicstrains fall outside the major clusters. Two of these, wE2 and wB3,associate at the periphery of the clusters representing the major groupsof novel alkaliphilic bacteria. Strain 79LN.4 and the related pair 72C.4and 80LN.4 are also non-clustering. Their inter-relationships are moredifficult to define but they probably represent new phenons presentlynot described.

S_(J)/UPGMA and S_(SM)/UPGMA Methods

The S_(J) coefficient is a useful adjunct to the S_(G) coefficient as itcan be used to detect phenons in the latter that are based on negativematches or distortions owing to undue weight being put on potentiallysubjective qualitative data. Consequently, the S_(J) coefficient isuseful for confirming the validity of clusters defined initially by theuse of the S_(G) coefficient. The Jaccard Coefficient is particularlyuseful in comparing biochemically unreactive organisms (Austin, B. andPriest, F. G., supra, p. 37). There may be doubts about theadmissability of matching negative character states (Sneath, P. H. A.,and Sokal, R. R., supra, p. 131) in which case the Simple MatchingCoefficient is a widely applied alternative.

In the main, all of the clusters (especially the clusters of the newbacteria) generated by the S_(G)/UPGMA method are recovered in thedendrograms produced by the S_(J)/UPGMA method (cophenetic correlation,0.795) (FIG. 2) and the S_(SM)/UPGMA method (cophenetic correlation,0.814) (FIG. 3). The main effect of these transformations is to gatherall the Bacillus strains in a single large cluster which further servesto emphasis the separation between the alkaliphilic Bacillus species andthe new alkaliphilic bacteria, and the uniqueness of the latter.

Chemotaxonomic Definition of the Clusters

Chemotaxonomy is the study of the chemical variations of cells inrelation to systematics. The analysis of chromosomal DNA, ribosomal RNA,proteins, cell walls and membranes, for example, can give valuableinsights into taxonomic relationships and may be used as a further toolto classify or to verify the taxonomy of microorganisms (Goodfellow, M.and Minnikin, D. E. in Chemical Methods in Bacterial Systematics, (eds.Goodfellow, M. and Minnikin, D. E.), Academic Press, London and Orlando,Fla. (1985), pp. 1-15). However, it is not always possible to decide aprior which type of chemical information will be most diagnostic for agiven classification. The amphipathic polar lipids, the majorrespiratory quinones, the fatty acids located in the bacterial membranesand the DNA base composition all have taxonomic significance for theclassification of various bacteria (Lechevalier, H. and Lechevalier, M.P., in Microbial Lipids, volume 1 (eds. Ratledge, C. and Wilkinson, S.G.) Academic Press, London and San Diego, Calif., (1988), pp. 869-902).

Polar Lipids

The extraction of polar lipids from bacteria and their analysis by twodimensional thin layer chromatography (2D-TLC) may yield patterns ofdiagnostic value. Stationary phase cells were extracted in 1:1 (v/v)CHCl₃:CH₃OH and examined by 2D-TLC as described by Ross, H. N. M.,Grant, W. D. and Harris, J. E., in Chemical Methods in BacterialSystematics, (eds. Goodfellow, M. and Minnikin, D. E.), Academic Press,London and Orlando, Fla., (1985), pp. 289-300. The types of lipidspresent on the chromatograms were visualized using a variety ofdifferential stains (Ross, H. N. M., et al., supra, p. 291, andTrincone, A., et al., Journal of General Microbiology, (1990), 136, pp.2327-2331). The identity of components were confirmed byco-chromatography.

The results of this analysis for representative strains of Gram-positivealkaliphiles are set out in Table 6. These show no clear polar lipidpattern which is distinct for any one cluster, although they do confirmthat phosphatidylethanolamine is a characteristic phospholipid of manyBacillus species, (O'Leary, . M. and Wilkinson, S. G., in MicrobialLipids, volume 1, supra, p. 157). However, we were surprised to findthat many of the bacteria contained one or several glycolipids,Glycolipids have not previously been demonstrated to be present inalkaliphilic bacteria (Krulwich, T. A., et al., CRC Critical Reviews inMicrobiology, (1988), 16, 15-36). Furthermore, as judged byco-chromatography of several strains, all glycolipid-containing strainscontained a glycolipid also found in Gram-negative alkaliphiles isolatedfrom soda lakes. Some of the other glycolipids appear to be common tocertain clusters of Gram-positive alkaliphiles. It is possibletherefore, that the chemical structures of the glycolipids will bechemotaxonomic markers for many obligate alkaliphiles in general and forspecific groups in particular.

TABLE 6 Polar Lipid Components of Gram-Positive Alkaliphilic BacteriaCLUSTER STRAIN PG DPG PGP PI PE GL 1 3E.1^(CT) + + + 3+71C.4 + + + + + + 81LN.4 + + + + + 2 69B.4 + 3+ RS11^(CT) + + 3+RS14 + + + + + + 3 wE1 + + + + + wN10 + + + + + wN16^(CT) + + + + + 413C.1 + + + + 3+ 23M.1 + + + + 3+ 14LN.1 + + + + 4+ 15LN.1^(CT) + + + +4+ — wE2 + + + + + 2+ 5 66B.4 + + + + + + RS17 + + + + + +AB49 + + + + + 6 RS7 + + + + 2+ RS15 + + + + 2+ — 72C.4 + + + + 3+ —80LN.4 + + + + 3+ PG phosphatidylglycerol; DPG diphosphatidylglycerol;PGP phosphatidylglycerolphosphate; PI phosphatidylinositol; PEphosphatidylethanolamine (ninhydrin positive aminolipid); GLunidentified glycolipid(s), α-naphthol positive (the number in thecolumn gives the number of positive spots on the TLC plate).

Isoprenoid Quinones

The isoprenoid or respiratory quinones are characteristic components ofthe plasma membrane of aerobic bacteria. There are two types;menaquinones and ubiquinones. The value of isoprenoid quinones astaxonomic criteria lies in the variation in the length of the polyprenylside-chain and the degree of saturation (Collins, M. D. and Jones, D.(1981), supra).

Dry stationary phase bacterial cells were extracted, using a modifiedprocedure of Collins, M. D. (in Chemical Methods in BacterialSystematics, supra, pp. 267-284), in 1:1 (v/v) CHCl₃:CH₃O at 50° C., for16 hours. The quinones were examined by reverse phase thin layerchromatography as described by Collins, M. D. (supra).

The results of quinone analyses of representative strains ofGram-positive alkaliphiles are illustrated in Table 7. However, these isno evidence to suggest that quinone composition is of value in thecircumscription of the clusters, although the data do serve to confirmthe status of these strains as Gram-positive. Furthermore, MK-7 as themajor isoprenologue of Bacillus species, including alkaliphilic strains,is also confirmed (Lechevalier, H. and Lechevalier, M. P., supra, p.881). Many of the novel, Gram-positive alkaliphilic bacteria of thepresent invention contain shorter molecules, especially MK-4 and MK-6,although no clear pattern emerges.

TABLE 7 Menaquinone Components of Gram-Positive Alkaliphilic BacteriaCLUS- TER STRAIN MENAQUINONE 1 3E.1^(CT) 3 4 71C.4 4 5 5H₂ 6 81LN.1 4 760E.4 3 4 8 wE4 4 5 2 69B.4 4 6 7 9 RS11^(CT) 7 8 3 wE1 6 7 wN10 6 wN126 7 wN16^(CT) 4 6 11 4 13C.1 4 6 7 9 23M.1 4 14LN.1 4 6 7 8 8H₂ 915LN.1^(CT) 5 6 7 8H₂ — wE2 7 — wB3 4 5 5 66B.4 4 6 7 RS17 4 7 8 AB49 76 RS7 4 5 7 8

Fatty Acids

The analysis of fatty acid profiles has had a significant impact onbacterial classification especially in the assignment of genera andspecies among Gram-positive bacteria and actinomycetes (Kroppenstedt, R.M., in Chemical Methods in Bacterial Systematics (eds. M. Goodfellow andD. E. Minnikin), Academic Press; London and Orlando, Fla., (1985), pp.173-199); Lechevalier, H. and Lechevalier, M. P., supra.

Freeze dried stationary phase cells (200-300 mg) were extracted for 16hours at 75° C. in toluene:methanol:conc. sulfuric acid (2.5 ml:2.5ml:0.2 ml) and after cooling, the lipids were partitioned into hexane(twice times 1 ml). Residual acid was removed using NH₄HCO₃. Lipidextracts were concentrated under O₂-free N₂, dissolved in 300 μl hexaneand applied to preparative silica gel plates (Merck F254, Type T). Theplates were developed in hexane: diethyl ether 85:15 (v/v) and the fattyacid methyl esters scraped off, extracted with hexane and concentratedunder a stream of O₂-free N₂.

The fatty acid methyl esters were dissolved in heptane and analysed bygas chromatography using a Packard model 430 chromatograph equipped withflame ionization detectors. The samples were divided by a samplesplitter and analyzed simultanously over two columns, namely, CP-SIL-88(Chrompack) (length 50 meter, internal diameter 0.22 mm) and Ultra-2(Hewlett Packard) (length 50 m, internal diameter 0.20 mm). The carriergas was nitrogen; the injection temperature 120° C.; temperaturegradient 2.5° C. per minute to 240° C. and isothermal at 240° C. for 30minutes. Faty acid methyl esters were assigned by reference to knownstandard mixtures. The identity of some peaks was confirmed by means ofgas chromatography-mass spectrometry using a Carlo Erba HRGC 5160 Megaseries gas chromatograph equipped with a CP-SIL-88 column (length 50meter, internal diameter 0.22 mm) with helium as carrier gas and directinjection into the source of a AND 403 mass spectrometer.

The fatty acid composition of the individual Gram-positive alkaliphilicbacteria is set out in Table 8. Table 9 shows the unique fatty acidprofiles of the individual clusters. Clusters 5 and 6 are typical forBacillus species with a predominance of branched C15:0 and C17:0 fattyacids. In spite of the homgeneity of cluster 4 shown by the numericaltaxonomy, fatty acid profiles clearly demonstrate that two subgroups ofbacteria (designated 4A and 4B) exist within this cluster. Theseprofiles are typical of some members of theCoryneform-Mycobacterium-Nocarbioform (CMN) groups of the actinomycetes(Bennan, P. J., in Microbial Lipids, supra. pp 203-298), a designationsupported by their characteristic cell habit, and the appearance ofdihydromenaquinones. Furthermore, the bright orange-yellow colors ofmany actinomycetes is caused by the accumulation of carotenoid pigments,often induced by light. Many of these carotenoids are unique andimportant taxonomically. Further chemotaxonomic markers for these groupsinclude a branched C19:0 fatty acid which is 10-methyloctadecanoic acid(tuberculostearic acid), an important criterion in the classification ofthe CMN group of bacteria. Branched, unsaturated fatty acids are alsofound in these bacteria. Branched C20:0 fatty acids, found in Clusters 3and 4 are components of some Gram-positive cocci. The control bacteriafor the coryneform group, namely Arthrobacter luteus (now reclassifiedas Oerskovia xanthionolytica) and Brevibacterium linens do notassociated closely with any of the clusters. However, the known obligatealkaliphiles among the CMN class of bacteria are poorly characterizedbut are clearly different from the novel Gram-positive alkaliphiles ofthe present invention.

TABLE 8 Fatty Acid Composition⁺ of Gram-Positive Alkaliphiles CLUSTERFATTY 1 2 3 4 5 6 UNCLUSTERED ACID 3E.1 81LN.4 69B.4 RS11 wE1 wN12 wN1623M.1 14LN.1 15LN.1 66B.4 AB49 RS8 RS16 wB3 72C.4 C11:0 t 0.2 — — t t —— — — — t — t — — C12:0 0.4 0.4 1.0 0.9 0.5 t 0.2 0.2 0.4 0.7 0.5 t 0.30.5 1.2 1.0 C14:0 3.4 3.2 5.9 6.0 4.3 1.5 5.3 1.8 3.5 5.1 3.1 2.9 3.93.6 7.4 6.9 C14:0 iso 0.7 — — — 0.7 0.2 — 0.5 — — 1.3 0.9 0.3 0.2 0.1 —C15:0 0.5 0.2 0.4 0.5 0.4 1.1 0.4 0.2 0.9 0.4 0.6 0.6 0.5 0.3 0.5 0.4C15:0 iso 3.2 0.8 0.3 0.3 1.9 7.6 0.2 1.4 — 0.2 13.1 13.2 18.9 3.1 0.50.4 C15:0 27.6 15.2 0.6 0.1 8.2 17.5 0.1 11.2 — 0.1 22.4 13.1 6.7 10.13.5 1.8 anteiso C16:0 17.6 21.2 28.1 30.6 26.2 22.9 30.8 9.8 29.4 28.415.8 22.3 25.5 23.0 30.6 30.7 C16:0 iso 5.1 0.5 0.1 — 0.7 1.1 — 13.0 — —6.4 6.1 1.4 0.4 1.0 0.7 C16:1 — — — — — — — — 3.1 t — — — — — — C16:1 br— — — — — — — 1.8 — — — — — — — — C17:0 0.4 0.4 0.7 0.7 0.6 1.0 0.6 3.83.3 0.8 0.7 0.6 0.4 0.5 0.6 0.6 C17:0 iso 1.4 0.6 — — 0.3 0.7 — 1.6 — —3.4 8.1 6.3 1.1 — — C17:0 14.2 13.4 — — 1.2 4.7 — 40.9 — — 10.4 12.3 4.15.4 3.1 1.4 anteiso C17:1 — — — — — — — — 2.6 — — 0.2 — — — — C18:0 12.922.2 31.1 31.2 28.0 28.5 30.5 6.7 18.7 30.1 11.3 10.7 16.8 28.5 25.028.4 C18:1 6.6 4.4 13.9 6.3 4.9 5.2 6.6 2.7 19.9 7.6 5.5 1.9 4.1 4.7 9.38.7 cis/trans C18:2 1.5 0.9 6.0 2.8 1.5* 2.3* 3.2* 1.1* 0.9* 4.1* 1.40.3 0.5 0.9 5.9* 5.0* unknown 0.5 — — 0.2 C19:0 br — — — — — — — — 3.20.4 — — — — — — C20:0 3.1 9.8 7.7 11.9 12.1 0.8 12.7 1.7 8.1 12.5 2.74.1 5.8 10.8 7.4 9.1 C22:0 1.5 6.2 4.2 8.2 8.2 5.1 8.8 0.9 5.7 9.0 1.62.7 3.8 6.8 3.9 5.5 C23:0 — — — — — — — 0.2 — — — — 0.3 — — — C24:0 t0.4 t 0.6 0.5 — 0.6 t 0.3 0.6 t 0.2 0.2 0.3 — — t = trace br =branched * = C18:2 and/or C20:0 br ⁺ = as % of total fatty acids

TABLE 9 Fatty Acid Profiles of the Clusters of Gram-PositiveAlkaliphiles CLUSTER 1 2 3 4A 4B 5 6 Predominant Fatty C15:0 anteisoC16:0 C16:0 C15:0 anteiso C16:0 C15:0 iso C15:0 br Acids (>10%) C16:0C18:0 C18:0 C16:0 C18:0 C15:0 anteiso C16:0 C17:0 anteiso C20:0 C17:0anteiso C16:0 C18:0 C18:0 C17:0 anteiso C18:0 n-saturated 40-65% 80-90%60-90% ≈25% 70-90% 35-45% 55-75% n-unsaturated <10% 10-20% <10%  ≈5%10-30% <10% ≈5% iso  1-10% <1% <10% ≈15%  <1% 20-30%  5-30% anteiso30-40% <1%  0-20% ≈50%  <1% 25-35% 10-15% total branched 30-50% <1% 0-30% ≈70%  <5% 50-60% 20-40% even carbon no. 50-70% >95%  70-99%≈40% >90% ≈50% 60-80% odd carbon no. 30-50% <5%  1-30% ≈60% <10% ≈50%20-40% additional markers C20:0 br C16:1 br C19:0 br C20:0 br C20:0 brbr = branched

DNA Base Composition

An important component of a taxonomic study is an analysis of thegenetic material—the nucleic acids. The composition of chromosomal DNAis unaffected by the growth conditions of the organism and anappropriate analysis may confirm or refute the taxonomic position of theorganism. Chromosomal DNA may be analyzed by the determination of thebase composition (G+C mol %) of individual strains. The guanine pluscytosine (G+C mol %) composition is constant for the chromosomal DNAfrom any given organism. Closely related organisms have similar G+Ccompositions. However, G+C results must be interpreted within thecontext of independent taxonomic data since similar G+C mol % of DNAsamples from different organisms does not in itself imply biologicalrelatedness.

DNA was extracted from cells grown to exponential phase in Medium A bythe choloroform:phenol method and was precipitated with ethanol. Basecomposition was determined by the thermal denaturation method (Marmur,J. and Doty, P. (1962), J. Mol. Biol., 3, 585-595) on a Phillips modelPV8764 spectrophotometer with temperature programming. A second methodinvolved HPLC analysis on a Beckman system gold using a Beckmanultrasphere ODS column and 0.04 M potassium dihydrogen phosphate plusacetonitrile (9+1, v/v) as eluent at a flow rate of 1.5 ml/min., aftertreatment of the DNA with nuclease P1 and alkaline phosphatase.

The results of these analyses are set out in Table 10. These results areconsistent with the group of the bacteria as defined by the numericaltaxonomy analysis. The G+C mol % values for the new alkaliphilicbacteria (clusters 1,3 and 4) cover a range of 29% (34.1-63.5 mol %).However, within these clusters the variation is less than 15 mol %,which confirms that the strains within a cluster are more closelyrelated to each other than to strains outside the cluster. Furthermore,it is evident that the new strains in clusters 1 and 4 with a high G+Ccontent (52.3-63.5 mol %) are clearly different from the known Bacillusstrains in clusters 2 (35.0-39.6 mol %), 5 (36.1-42.8 mol %) and 6(36.5-43.6 mol %). The new strains of clusters 3 (G+C=34.1-46.3 mol %)are clearly differentiated from the bacilli on the basis of otherchemotaxonomic data.

TABLE 10 DNA Base Composition of Gram-Positive Alkaliphilic Bacteria G +C mol % Cluster Strain HPLC T_(M) Literature¹⁾ 1 3E.1^(CT) 52.3 wE4 63.12 RS11^(CT) 35.0 35.2 RS14 39.6 RS13 37.2 3 wE1 49.3 wN10 34.1 wN12 40.136.6 wN16^(CT) 46.0 40.2 4 23M.1 63.0 15LN.1^(CT) 63.5 5 66B.4 42.1RS10^(CT) 39.5 RS17 42.5 AB49 42.1 42.8 AB42 36.1 6 RS7 43.2 RS8^(CT)39.9 43.6 RS15 43.5 RS16 36.5 ¹⁾Fritze, D., Flossdorf, D. and Claus, D.(1990) Int. J. Systematic Bacteriology, 40, 92-97.

Determination of Representative Strains

The centroid of each individual cluster generated by the S_(G)/UPGMAmethod was computed using the RGROUPS program in TAXPAK. The centroid ofa cluster of points representing real organisms projected intohyperspace represents a hypothetical average organism. The centroidseldom, if ever, represents a real organism. Therefore, the Euclideandistances of each of the members of the cluster from the centroid of thecluster were calculated in order to establish which organism was closestto the hypothetical average organism. The organism closest to thecentroid was designated the “centrotype organism” (indicated with thesuperscript “CT”).

The centrotype organism can be thought of as the “Type Strain” whichmost closely represents the essential and discriminating features ofeach particular cluster. The centrotype strains are recorded in Table11.

TABLE 11 Centrotype Strains Mean Euclidean Centrotype Distance ofEuclidean Number of Strains Distance Cluster Strains in from Standardfrom Number Cluster Centroid Deviation Strain Centroid 1 5 6.24 0.653E.1 4.45 2 5 6.02 0.23 RS11 4.53 3 4 6.81 0.96 wN16 4.26 4 4 6.40 0.5715LN.1 4.40 5 6 6.44 0.27 RS10 5.01 6 4 6.50 0.72 RS8 4.06

A description of each of the centrotype organisms from the clusterscontaining the novel bacteria has been made so as to be able todistinguish these organisms from all other bacteria previously known anddescribed. In addition, the minimum number of discriminatory tests todefine each cluster has been computed so that it may be clearly seenthat the clusters containing these novel bacteria can be easilydistinguished from each other and from all other known bacteria.

Description of Centrotype Strains

Strain 3E.1^(GT) (Cluster 1)

An aerobic, Gram-positive coccoid bacterium. The cells are almostspherical, 05.-1.5 μm, usually in pairs or occasionally tetrads, formingshort chains of up to 6 cells, or in irregular clusters.

Motility not observed.

Obligate alkaliphile, grows optimally at about pH 10.

On alkaline-agar, (Medium A) forms opaque, matte orange-coloredcolonies. The color development is influenced by light, beginning ascream developing through yellow to orange. The colonies are circular,convex, entire, 1-2 mm in diameter.

In alkaline-broth (Medium A), growth (37° C.) is moderate, evenlyturbid, with the formation of a sediment but no surface pellicle.

Optimum temperature for growth is about 30° C. Grows slowly at 15° C.and 40° C. No growth at 45°° C.

KOH test negative Aminopeptidase test negative Oxidase reaction negativeCatalase reaction positive NaCl tolerance 0% to 12%. No growth at 15%.Hydrolysis of Gelatin positive Hydrolysis of Starch weak positive. Majorpolar lipid phosphatidylglycerol, components diphosphatidylglycerol,phosphatidylglycerolphosphate. Three glycolipid (α-naphthol positive)components present. Major menaquinones MK-3, MK-4. Major fatty acidsC15:0 anteiso, C16:0, C17:0 anteiso, C18:0. G + C 52.3 mol % (HPLC)

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones. Growth on simple sugars and organic acids very restricted.Growth is stimulated by glucose; acetate, some amino acids andpyrimidine nucleotides.

Strain wN16^(CT) (Cluster 3)

An aerobic, Gram-positive bacterium. The cells are short, thick,slightly irregular rods, 1.5-2.5 μm×0.75-1.0 μm, occuring singly or inpairs, sometimes in short chains of up to 4 cells.

No spores observed. Motility not observed.

Obligate alkaliphile; no growth below pH 8.

On alkaline-agar, (Medium A) forms smooth, cream-yellow coloredcolonies. The colonies are small, about 1 mm in diameter, circular,entire and convex.

In alkaline-broth (Medium A), growth (37° C.) is moderate, evenlyturbid, forming a sediment but not surface pellicle.

Grows well between 20° C. and 40° C. Grows slowly at 10° C.; no growthat 45° C.

KOH test negative Aminopeptidase test negative Oxidase reaction negativeCatalase reaction negative NaCl tolerance 0% to 10%. Hydrolysis ofGelatin positive Hydrolysis of Starch positive. Major polar lipidphosphatidylglycerol, components diphosphatidylglycerol,phosphatidylglycerolphosphate, phosphatidylinositol,phosphatidylethanolamine. Major menaquinones MK-4, MK-6, MK-11. Majorfatty acids C16:0, C18:0, C20:0 (fatty acids with even carbon numberscomprise > 95%, branched fatty acids < 1%). G + C 40.2 mol % (T_(M)) -46.0 mol % (HPLC)

Chemoorganotroph, Grows on complex substrates such as yeast extract, arange of sugars, amino- and organic acids.

Strain 15LN.1^(CT) (Cluster 4)

An aerobic, Gram-positive bacterium. The cells are initially irregular,spherical or elongated, sometimes wedge-shaped, 1.5-2.0 μm×0.75-1 μm,developing into short, thick, slightly curved rods. 1-3 μm×0.5-1 μm. Thecells often occur in pairs. Due to the characteristic snapping form ofcell division, the cells are frequently found at an angle forming aV-shaped, or in clusters of cells with a pallisade appearance.

No spores observed. No motility observed.

Obligate alkaliphile; no growth below pH 7.5, optimum pH≅9-10.

On alkaline-agar, (Medium A) forms brightly colored, smooth, glisteningcolonies, initially orange developing into red. Color development isinfluenced by light. The colonies are circular, convex, entire, opaque,1-2 mm in diameter.

In alkaline-broth (Medium A), growth (37° C.) is slight to moderate,flocculent, with the formation of sediment and a surface ring orpellicle.

Grows well between 20° C. and 40° C. Grows slowly at 45° C. and 10° C.No growth at 50° C.

KOH test negative Aminopeptidase test negative Oxidase reaction negativeCatalase reaction positive NaCl tolerance 0% to 8%. Hydrolysis ofGelatin negative Hydrolysis of Starch positive. Major polar lipidphosphatidylglycerol, components diphosphatidylglycerol,phosphatidylglycerolphosphate, phosphatidylinositol. Four glycolipid(α-naphthol positive) components present. Major menaquinones MK-5, MK-6,MK-7, MK-8(H₂) Major fatty acids C16:0, C18:0, C20:0 (fatty acids witheven carbon numbers comprise > 95%, branched fatty acids < 1%). G + C63.5 mol % (HPLC)

Chemoorganotroph. Grows on complex substrates such as yeast extract andpeptones. Growth on simple sugars very restricted. Growth is stimulatedby amino acids and fatty acids.

Non-clustering Strains

The strains which do no fall into the clusters defined here are alsonovel bacteria not previously known or described. These strains, codedwE2, wB3, 72C.4, 79LN.4 and 80LN.4, may represent rarer varieties ofalkaliphilic bacteria. Some of these strains, such as wE2 and wB3 mayrepresent intermediate forms, falling between closely related (andclosely oriented in hyperspace) clusters as defined here. The otherstrains. 72C.4, 79LN.4 and 80LN.4, are probably members of clusters ofbacteria representing new genera or species at present not defined. Adescription of these “non-clustering” strains has been made so as to beable to distinguish these organisms from all other bacteria previouslyknown and described.

Strain wE2

An aerobic, Gram-positive bacterium. The cells are irregular; mainlyoval coccoid cells, 1-2 μm×0.5-1 μm, or very short rods, occasionally inpairs, or slightly curved short rods. Due to the characteristic snappingform of cell division, the cells are frequently found at an angleforming a V-shape. Alkaliphilic bacterium wE2 was deposited underaccession number CBS 112.95 and received on Jan. 20, 1995 by theCentraalbureau voor Schimmelcultures, having the address Oosterstraat 1,P.O. Box 273, 3740 AG BAARN, The Netherlands.

No spores observed. No motility observed.

Obligate alkaliphile; no growth below pH 8.

On alkaline-agar, (Medium A) forms opaque, orange colored, punctiform orcircular colonies, with a convex or domed elevation and entire margin,up to 1 mm in diameter.

In alkaline-broth, (Medium A) growth (37° C.) is slow, slight tomoderate, flocculent turbidity, with the formation of a sediment andsurface ring.

Temperature: grows optimally at above 30° C., slowly at 10° C. No growthat 40° C.

KOH test negative Aminopeptidase test negative Oxidase reaction negativeCatalase reaction positive NaCl tolerance 0% to 10%. No growth at 12%.Hydrolysis of Gelatin negative Hydrolysis of Starch negative Major polarlipid phosphatidylglycerol, components diphosphatidylglycerol,phosphatidylglycerolphosphate, phosphatidylinositol,phosphatidylethanolamine. Two glycolipids (α-naphthol positive)components present. Major menaquinones MK-7

Chemoorganotroph. Grows on complex substrates such as yeast extract,peptones and carbohydrates (dextrin). Growth is stimulated by a varietyof sugars, organic-, fatty- and amino-acids.

Strain wE2 appears to be an intermediate form related to Cluster 4.

Strain wB3

An aerobic, Gram-positive bacterium. The cells are short, straight orslightly curved rods, 1-2.5 μm×0.5 μm, sometimes in pairs. Due to thecharacteristic snapping form of cell division, the cells are frequentlyfound at an angle forming a V-shape. Alcaliphilic baterium wB3 wasdeposited under accession number CBS 111.95 and received on Jan. 20,1995 by the Centraalbureau voor Schimmelcultures, having the addressOosterstraat 1, P.O. Box 273, 2740 AG BAARN, The Netherlands.

Motility not observed. No spores observed.

Obligate alkaliphile; no growth below pH 8. On alkaline-agar, (Medium A)forms opaque yellow-ochre, circular, convex, entire colonies, 2 mm indiameter.

In alkaline-broth, (Medium A) growth (37° C.) is slight to moderate withan even turbidity and the formation of a sediment, but no surfacepellicle.

Temperature range for growth: 10° C. to 40° C. No growth at 45° C.

KOH test negative Aminopeptidase test negative Oxidase reaction negativeCatalase reaction positive NaCl tolerance 0% to 12%. No growth at 15%.Hydrolysis of Gelatin positive Hydrolysis of Starch negative Majormenaquinones MK-4, MK-5. Major fatty acids C16:0, C18:0 (fatty acidswith even carbon numbers > 90%, branched fatty acids < 10%).

Chemoorganotroph. Grows on complex substrates such as yeast extract.Growth on simple substrates (sugars, etc.) very restricted. Growth isstimulated by acetate and glucose. Strain wB3 appears to be anintermediate form related to Cluster 1.

Strain 79LN.4

An aerobic, motile, Gram-positive bacterium. The cells are straight orslightly curved rods, 1.5-5 μm×0.5-1 μm, often in pairs, sometimes inshort chains of 2 to 4 cells. Alcaliphilic bacterium 79LN.4 wasdeposited with accession number CBS 109.95 and received on Jan. 20, 1995with the Centraalbureau voor Schimmelcultures, having the addressOosterstraat 1, P.O. Box 273, 3740 AG BAARN, The Netherlands.

No spores observed. Motility not observed.

Obligate alkaliphile, no growth below pH 7.5.

On alkaline-agar, (Medium A) forms opaque, cream colored colonies, 2 mmin diameter. The colonies are circular, umbonate in elevation, with anentire margin becoming undulate with age.

In alkaline-broth, (Medium A) growth (37° C.) is moderate to heavy,evenly turbid, with the formation of a sediment and eventually a surfacering.

Temperature range: grows well at 20° C. to 40° C. Grows slowly at 10° C.and 45° C. No growth at 50° C.

KOH test negative Aminopeptidase test negative Oxidase reaction weakpositive Catalase reaction positive NaCl tolerance 0% to 15% Hydrolysisof Gelatin positive Hydrolysis of Starch positive

Chemoorganotroph. Grows well on complex substrates, simple sugars,organic-, amino- and fatty acids, and pyrimidine nucleotides.

Strain 72C.4

An aerobic, Gram-positive bacterium. The cells appear to have a distinctcoccus-rod development cycle. Initially the cells are spherical orirregular coccobacillery in form which develop into short rods, 1-2.5μm×0.5-0.75 μm. Eventually some longer forms, 3-4 μm×1 μm appear. Thecells occur occasionally in pairs. Due to the characteristic snappingform of cell division, the cells are frequently found at an angleforming a V-shape. Alcaliphilic bacterium 72C.4 was deposited havingaccession number CBS 108.95 and received on Jan. 20, 1995 with theCentraalbureau voor Schimmelcultures, having the address Oosterstraat 1,P.O. Box 273,3740 AG BAARN, The Netherlands.

No motility observed. No spores observed.

Obligate alkaliphile; no growth below pH 8.

On alkaline-agar, (Medium A) forms circular, convex, entire, opaquecolonies, 1-2 mm in diameter. The colony color is initially orangedeveloping with age and the influence of light into a deep salmon pink.

In alkaline-broth, (Medium A) growth (37° C.) is moderate, evenlyturbid, with the formation of a sediment but no surface pellicle.

Temperature range: grows well at 20° C. to 37° C. Grows slowly at 10° C.No growth at 40° C.

KOH test negative Aminopeptidase test positive Oxidase reaction negativeCatalase reaction positive NaCl tolerance 0% to 12% Hydrolysis ofGelatin positive Hydrolysis of Starch negative Major polar lipidphosphatidylglycerol, components diphosphatidylglycerol,phosphatidylglycerolphosphate, phosphatidylinositol. Three glycolipid(α-naphthol positive) components present. Major fatty acids C16:0,C18:0, (fatty acids with even carbon numbers > 95%, branched fatty acids< 5%).

Chemoorganotroph. Grows well on complex substrates (e.g. yeast extract)and a variety of sugars, organic acids and amino acids.

Strain 80LN.4

An aerobic, Gram-positive bacterium. The cells are nearly spherical orcoccobacillery in form, developing into very short rods, 1-2 μm×0.5-0.75μm. Occasionally longer forms occur. The cells occur occasionally inpairs.

No motility observed. No spores observed.

Obligate alkaliphile, no growth at pH 8.

On alkaline-agar, (Medium A) forms circular, convex to umbonate, entire,opaque colonies, 1-2 mm in diameter. The colony color is initiallyorange developing with age and the influence of light into a deep salmonpink.

In alkaline-broth, (Medium A) growth (37° C.) is moderate, evenlyturbid, with the formation of sediment and surface pellicle.

Temperature range: grows well at 20° C. to 37° C. Grows slowly at 10° C.No growth at 40° C.

KOH test negative Aminopeptidase test positive Oxidase reaction negativeCatalase reaction positive NaCl tolerance 0% to 12%. Grows weakly at15%. Hydrolysis of Gelatin positive Hydrolysis of Starch negative Majorpolar lipid phosphatidylglycerol, components diphosphatidylglycerol,phosphatidylglycerolphosphate, phosphatidylinositol. Three glycolipid(α-naphthol positive) components present.

Chemoorganotroph. Grows well on complex substrates (e.g. yeast extract)and a variety of sugars, organic acids and amino acids.

Cluster Definition by the Calculation of the Minimum Number ofDiscriminatory Tests, and the Construction of a Probability Matrix forthe Identification of Gram-positive Alkaliphiles

One of the purposes of a numerical classification study is to use thephenetic data, which define the clusters at a selected similarity level,for the assignment or identification of unknown strains. Theclassification test data can be used to determine the minimum set oftests which are required to define the clusters at the 79% (S_(G))similarity level, and to identify those characters which are mostdiagnostic (predictive) for the individual clusters. In other words, theminimum number of tests which are required to assign an unknown organismto a pre-determined cluster with a high degree of predictability.

From the minimum discriminatory tests, a probability matrix can beconstructed for the identification of unknown strains. The analysis isachieved by using a combination of the CHARSEP and DIACHAR programs inTAXPAK, supplemented by the MCHOICE program (not on TAXPAK, butavailable by Data-Mail from the University of Leicester, U.K.). Anevaluation of the identification matrix is provided by using theMOSTTYP, OVERMAT and MATIDEN programs. Practical examples of the use ofthese programs for the probabilistic identification of bacteria havebeen published by Williams, S.T., et al., (1983), Journal of GeneralMicrobiology, 129, 1815-1830; and Priest, F. G. and Alexander, B.,(1988), Journal of General Microbiology, 134, 3011-3018; ibid, (1990),136, 367-376.

A “n×t” table was constructed using the two-state characters from thetest data. In other words, using characters 6 to 11 and 14 to 200(Appendix C) scored in binary notation (positive=1, negative=0).

The data matrix is first examined using the CHARSEP program whichcalculates separation indices and thus the diagnostic value of theindividual characters for discriminating between the clusters.Character-states (tests) with a VSP index [(4 times variance) timesstrain potential] greater than 25% (Sneath, P. H. A., (1979), Computersand Geosciences, 5, 349-357) are accepted, characters with a lowdiagnostic value (VSP <25%) are rejected. A preference is made forcharacters with the highest VSP indices, provided that the criteria inthe DIACHAR and MCHOICE programs are also met. In this example, 63 testshave a VSP index >25%, and 16 of the 32 characters finally chosen have aVSP index >50% (Table 12).

The data matrix is next re-examined by means of the DIACHAR program,which determines the most diagnostic character states of each of theclusters. The number of character states was set as 12. This resultallows the choice of mutually exclusive character states between theclusters. As many of these tests as possible are retained in the finalidentification matrix of minimum discriminatory tests; in this examplebetween 4 and 10 diagnostic characters per cluster. The remaining,unused tests are also noted and may be applied as additional tests forthe confirmation of identification (Table 13).

The MCHOICE program ranks the tests in groups which can be displayed inthe form of a dendrogram using the MDEND sub-routine. The groupsidentify tests with similar discriminatory value, thus allowing therejection of tests which fail to make a significant discrimination aswell as allowing choices to be made between tests of equal or verysimilar diagnostic value.

Table 14 shows the set of 32 tests which is the minimum number requiredto define the clusters and which can be used for the assignment ofunknown strains. In addition, Table 14 shows the identification matrixwhich consists of the percentage of positive characters which define theclusters on the basis of the 32 minimum discriminatory tests. This iscomputed by the IDMAT program.

TABLE 12 Separation Values of Characters used for the MinimumDiscriminatory Tests CHARACTER VSP Index  [10] Gelatin 30.6  [14]Fumarate 35.4  [15] Fructose 35.2  [19] Galactose 34.8  [24]N-acetylglucosamine 58.1  [27] D-saccharose 74.1  [28] Maltose 70.7 [32] Acetate 56.8  [36] D-glucose 63.1  [37] Salicin 51.3  [38]D-melibiose 45.0  [42] Propionate 72.4  [44] Valerate 31.7  [48]Glycogen 85.3  [50] L-serine 38.1  [63] Chymotrypsin 44.4  [70]β-glucosidase 67.1  [74] Serine 58.6  [77] Arginine 65.3  [80]Methionine 54.0  [90] Penicillin G 55.9  [94] Methicillin 56.5  [96]Streptomycin 28.5  [97] Tetracyclin 51.8 [105] Bacitracin 32.8 [112]N-acetyl-D-glucosamine 32.8 [116] Cellobiose 45.0 [137] Turanose 55.5[139] Methyl pyruvate 41.1 [140] Mono-methylsuccinate 39.1 [192]Thymidine 34.9 [197] Glycerol 34.0

TABLE 13 Discriminatory Tests for Each of the Six Clusters (S_(G))Positive Negative Cluster 1: matte orange colored circular colonies;coccoid cells.  [10] Gelatin hydrolysis  [24] N-acetylglucosamine  [90]Penicillin G  [27] D-saccharose  [94] Methicillin  [37] Salicin [105]Bacitracin  [38] D-melibiose  [42] Propionate  [48] Glycogen  [52] 3hydroxybutyrate  [74] Serine  [77] Arginine  [80] Methionine  [83]Valine [116] Cellobiose Cluster 2:  [10] Gelatin hydrolysis  [24]N-acetylglucosamine  [15] Fructose  [42] Propionate  [28] Maltose  [44]Valerate  [37] Salicin  [50] L-serine  [70] β-glucosidase  [89]Trimethoprim  [74] Serine [123] m-inositol  [80] Methionine [197]Glycerol  [86] Ampicillin  [94] Methicillin  [96] Streptomycin [105]Bacitracin [137] Turanose Cluster 3: yellow colonies; rod-shaped cellsin chains.  [10] Gelatin hydrolysis  [28] Maltose  [19] Galactose  [32]Acetate  [70] β-glucosidase  [36] D-glucose  [77] Arginine  [38]D-melibiose [105] Bacitracin  [42] Propionate [112]N-acetyl-D-glucosamine  [44] Valerate [116] Cellobiose  [48] Glycogen[190] Inosine  [74] Serine [191] Uridine  [86] Ampicillin  [90]Penicillin G  [94] Methicillin  [97] Tetracyclin [139] Methyl pyruvate[140] Mono-methylsuccinate [155] α-ketobutyric acid Cluster 4:glistening bright orange/red colonies; cells coccobacillery  [77]Arginine  [10] Gelatin hydrolysis  [80] Methionine  [14] Fumarate  [82]Glycine  [15] Fructose  [83] Valine  [19] Galactose  [94] Methicillin [27] D-saccharose  [97] Tetracyclin  [38] D-melibiose [105] Bacitracin [48] Glycogen [106] α-cyclodextrin  [50] L-serine [109] Tween 40  [70]β-glucosidase [139] Methyl pyruvate [112] N-acetyl-D-glucosamine [140]Mono-methylsuccinate [116] Cellobiose [151] β-hydroxybutyric acid [134]D-sorbitol [165] Bromo-succinic acid [136] D-trehalose [137] TuranoseCluster 5: beige or dull cream colonies; straight rod-shaped cells. [10] Gelatin hydrolysis [140] Mono-methylsuccinate  [24]N-acetylglucosamine [157] α-ketovaleric acid  [27] D-saccharose  [28]Maltose  [32] Acetate  [36] D-glucose  [48] Glycogen  [63] Chymotrypsin [86] Ampicillin  [94] Methicillin  [97] Tetracyclin [137] TuranoseCluster 6: cream colonies; straight rod-shaped cells.  [10] Gelatinhydrolysis  [19] Galactose  [15] Fructose  [63] Chymotrypsin  [24]N-acetylglucosamine  [74] Serine  [27] D-saccharose  [75] Proline  [28]Maltose  [77] Arginine  [32] Acetate  [96] Streptomycin  [36] D-glucose [99] Oleandomycin  [37] Salicin [105] Bacitracin  [38] D-melibiose[116] Cellobiose  [42] Propionate [140] Mono-methylsuccinate  [45]Citrate [192] Thymidine  [48] Glycogen  [50] L-serine  [66]α-galactosidase  [67] β-galactosidase  [68] β-glucuronidase  [70]β-glucosidase [123] m-inositol [137] Turanose [197] Glycerol Note: Thenumbers in square brackets proceeding the character state refers to thecharacter states and unit tests in Appendices B and C.

TABLE 14 Identification Matrix: Percentage of Positive DiscriminatoryCharacters which Define the Clusters of Gram-Positive AlkaliphilicBacteria at the 79% Level (S_(G)) CLUSTER TEST 1 2 3 4 5 6  [10] Gelatin100 100 100 0 100 100  [14] Fumarate 20 25 75 0 83 50  [15] Fructose 60100 75 0 83 100  [19] Galactose 20 25 100 0 17 0  [24]N-acetylglucosamine 0 0 50 25 100 100  [27] D-saccharose 0 75 25 0 100100  [28] Maltose 20 100 0 25 100 100  [32] Acetate 20 25 0 75 100 100 [36] D-glucose 20 75 0 25 100 100  [37] Salicin 0 100 50 25 67 100 [38] D-melibiose 0 50 0 0 50 100  [42] Propionate 0 0 0 75 83 100  [44]Valerate 20 0 0 50 83 50  [48] Glycogen 0 75 0 0 100 100  [50] L-serine40 0 25 0 17 100  [63] Chymotrypsin 40 25 75 25 100 0  [70]β-glucosidase 20 100 100 0 33 100  [74] Serine 0 100 0 75 50 0  [77]Arginine 0 50 100 100 33 0  [80] Methionine 0 100 nc 100 33 25  [90]Penicillin G 100 75 0 50 83 0  [94] Methicillin 100 100 0 100 100 25 [96] Streptomycin 40 100 50 75 67 0  [97] Tetracyclin 80 75 0 100 10025 [105] Bacitracin 100 100 100 100 83 0 [112] N-acetyl-D- 40 25 100 050 50 glucosamine [116] Cellobiose 0 50 100 0 50 0 [137] Turanose 40 10025 0 100 100 [139] Methyl pyruvate 60 75 0 100 33 75 [140]Mono-methylsuccinate 40 25 0 100 0 0 [192] Thymidine 80 25 50 50 83 0[197] Glycerol 80 0 50 25 33 100 nc = not computed

Evaluation of the Discriminatory Tests and Assessment of the Reliabilityof Identification

The evaluation of the discriminatory tests has two aspects. Firstly, thevalidity of the tests can be analysed using practical examples, whichcan be further evaluated using statistical theory, or the tests can bedirectly subjected to theoretical assessment using statistical methods.

Illustration 1 A Practical Evaluation of the Discriminatory Tests

Many workers assess the accuracy of the discriminatory tests only byredetermining the character states of selected cluster representatives.This approach has been used here for the centrotype strains (see below).A far more stringent approach which is seldom applied, is to examine allthe strains which were used in the original numerical taxonomicanalysis. When subjected to cluster analysis using only the dataacquired from the derived set of minimum discriminatory tests, thereconstructed dendrogram can be compared with the original. Using onlythe 32 discriminatory tests previously described (Table 14), the data(two-state, binary form) for all 20 of the new Gram-positivealkaliphilic bacteria and 13 known alkaliphilic Bacillus speciesisolated by Japanese workers, were subjected to cluster analysis by theS_(G)/UPGMA method (equivalent in this case to the S_(SM)/UPGMA method).The reconstructed dendrogram is reproduced in FIG. 4. This reconstructeddendrogram compares very favorably with the original dendrograms (FIGS.1 and 2).

Although there has been some rearrangement of position of the clusters,their composition is largely unchanged and there is a clear separationbetween the clusters of novel alkaliphilic bacteria of the presentinvention and the alkaliphilic Bacillus species.

This evidence, together with the statistical data provided by thenumerical taxonomic analysis and the chemotaxonomic data, indicates arobust classification which identifies three major groups of novelGram-positive alkaliphilic bacteria.

Illustration 2 A Theoretical Evaluation of the Discriminatory Tests

The significance of the apparent clear cluster separation obtained inIllustration 1 (above) can be evaluated using the OVERMAT program whichassesses cluster overlap between taxa in an identification matrix. Thisprogram examines the matrix constructed from the percentage positivevalues for the selected character states against a critical overlapvalue by considering the clusters defined by the coordinates of thecentroid and cluster radius (twice root mean square of the distances ofthe strains of the cluster from the centroid). If there is significantoverlap between the clusters, unknown strains may not identify withsufficient confidence to any one of them (Sneath, P. H. A. and Sokal, R.R., supra, p. 394-400). At a chosen critical overlap value of 2.5%(which is a more stringent condition than is used by most workers: seePriest, F. G. and Alexander, B., (1988), supra; and Williams, S. T. etal. (supra), there was no significant overlap (99% confidence level)between most of the clusters (Table 15). Even at a 1% critical overlapvalue there was no significant cluster overlap (Table 16) except betweenCluster 2 and Cluster 5, but since these both represent Bacillus strainsthis is not considered to have any practical significance for thecorrect identification of the new Gram-positive alkaliphiles.

TABLE 15 Percentage Probability that Cluster Overlap is < 2.5% CLUSTER 12 3 4 5 6 1 2 99 3 99 99 4 95 99 99 5 99 95 99 99 6 99 99 99 99 99

TABLE 15 Percentage Probability that Cluster Overlap is < 2.5% CLUSTER 12 3 4 5 6 1 2 99 3 99 99 4 95 99 99 5 99 95 99 99 6 99 99 99 99 99

Illustration 3 A Theoretical Assessment of the Reliability ofIdentification

The hypothetical median organism (HMO) is another estimate of the“average” organism in a cluster (Sneath, P. H. A. and Sokal, R. R.,supra, pp. 195, et seq.). A HMO is not a real strain but a hypotheticalorganism possessing the most common state for each character. TheMOSTTYP program calculates HMO's for each cluster in the identificationmatrix and then attempts to identify them. In other words, MOSTTYP is aprogram to evaluate an identification matrix by calculatingidentification scores of the most typical strains against the clusters.A good identification matrix should give a high probability of a HMObeing reassigned to its own cluster. The results of this analysis werevery satisfactory. Each HMO was reassigned to its original cluster withWillcox probabilities of 1.000 (Willcox, W. R. et al., (1973) Journal ofGeneral Microbiology, 77, 317-330). The taxonomic distances were all lowand the standard errors of the taxonomic distance were all negative,indicating that the HMO's were all closer to the centroid of the clusterthan the average for the cluster (Table 17).

TABLE 17 Identification Scores for the Hypothetical Median Organism ofeach cluster provided by the MOSTTYP Program Identification ScoreWillcox Taxonomic Standard Error of CLUSTER Probability DistanceTaxonomic Distance 1 1.000 0.229 −3.086 2 1.000 0.221 −3.200 3 1.0000.242 −2.288 4 1.000 0.207 −3.090 5 1.000 0.251 −2.701 6 1.000 0.177−2.690

Illustration 4 A Practical Evaluation of Identification Score

Identification of strains using the minimum set of discriminatory testsis achieved using the MATIDEN program in TAXPAK. The program comparespresence-absence data for an unknown strain against each cluster in turnin an identification matrix of percentage positive characters.Identification coefficients are computed, namely Willcox probability,Taxonomic Distance and the Standard Error of the Taxonomic Distance. Theresults are displayed, showing the identification scores to the bestcluster and to the two next best alternative clusters. Additionally, theatypical results (“characters against”) are recorded. In an analysisusing data from real strains, the centrotypes were reassigned to theiroriginal clusters with Willcox probabilities of 1.000 (Table 18). Thetaxonomic distances were low, generally in the same range as the HMO's.The standard errors of the taxonomic distance were all negativeindicating that the centrotypes were closer to the centroid of thecluster than the average for the cluster. The exception was Bacillusreference strain RS10 but this was well within the acceptable limits of+3.0 (Sneath, P. H. A. (1979), pp. 195-213).

TABLE 18 Identification Scores for the Centrotype Organisms of EachCluster Provided by the MATIDEN Program Identification Score Clus-Assigned Willcox Taxonomic Standard ter Strain to Cluster ProbabilityDistance (D) Error of D 1 3E.1^(CT) 1 1.000 0.289 −1.088 2 RS11^(CT) 21.000 0.273 −0.584 3 WN16^(CT) 3 1.000 0.229 −1.767 4 15LN.1^(CT) 41.000 0.217 −0.519 5 RS10^(CT) 5 1.000 0.421 −1.678 6 RS8^(CT) 6 1.0000.221 −2.468

Illustration 5 Identification of an Unknown Isolate

The identification matrix was assessed for the ability to assign anunknown Gram-positive alkaliphile to the clusters defined herein. Thecriteria for a successful identification were:

(a) a bacterium isolated from a similar habitat to, but geographicallyseparate from, the East African soda lakes;

(b) a Willcox probability greater than 0.95 and low values for taxonomicdistance and its standard error (<3);

(c) an identification score to the best cluster significantly betterthan those against the two next best alternatives;

(d) “characters against” the best cluster should be zero or few innumber.

An unknown microorganism may be examined using the minimum tests listedin Table 14. The character states are determined and identificationscores obtained using the MATIDEN program. This program compares thecharacter states of the unknown with the identification matrixdetermined for all of the predetermined clusters, computes the bestmatch and assigns the unknown to the most appropriate cluster.

A Willcox probability is calculated to determine the acceptability ofidentification. Willcox probabilities of 0.85 and 0.95 have beenaccepted as criteria for a successful identification (Williams, S. T.,et al., (1983), supra; Priest, F. G. and Austin, B., (1988), supra). Thetaxonomic distance of the unknown from the cluster centroid iscalculated and may be compared to the radius of the cluster. Thestandard error of the taxonomic distance should be less than the uppervalue of +3.0 suggested by Sneath, P. H. A. ((1979), pp. 195-213).Moreover, physical characteristics, additional biochemical data andchemotaxomomic markers may be used to further confirm the identity ofthe unknown in a particular cluster.

Production and Application of Alkalitolerant Enzymes

The alkaliphilic microorganisms of the present invention produce avariety of enzymes. These enzymes are capable of performing theirfunctions at an extremely high pH, making them uniquely suited for theirapplication in a variety of processes requiring such enzymatic activityin high pH environments or reaction conditions.

Examples of the various applications for alkalitolerant enzymes are indetergent compositions, leather tanning, food treatment, waste treatmentand in the textile industry. These enzymes may also be used forbiotransformations, especially in the preparation of pure enantiomers.

The alkaliphiles may easily be screened for the production ofalkalitolerant enzymes having lipolytic, proteolytic and/orstarch-degrading activity using the methods described in Appendix B.

The broth in which alkaliphilic bacteria are cultured typically containsone or more types of enzymatic activity. The broth containing the enzymeor enzymes may be used directly in the desired process after the removalof the bacteria therefrom by means of centrifugation or filtration, forexample.

If desired, the culture filtrate may be concentrated by freeze drying,before or after dialysis, or by ultrafiltration. The enzymes may also berecovered by precipitation and filtration. Alternatively, the enzyme orenzymes contained in the broth may be isolated and purified bychromatographic means or by gel electrophoresis, for example, beforebeing applied to the desired process.

The genes encoding alkalitolerant enzymes of interest may be cloned andexpressed in organisms capable of expressing the desired enzyme in apure or easily recoverable form.

In one embodiment, the enzymatic preparation may be used in wash teststo determine the efficacy of the enzymatic activity.

Enzyme preparations from the alkaliphilic bacteria may be tested in aspecially developed mini-wash test using cotton swatches soiled, forexample, with protein-, lipid- and/or starch-containing components.Prior to the wash test, the swatches can be pre-treated with a solutioncontaining an anionic surfactant, sodium perborate and a bleachactivator (TAED). After this treatment, the test swatches are rinsed inrunning demineralized water and air-dried. This treatment results in thefixation of the soil, making its removal more difficult.

The washing tests may be performed using a defined detergent compositionplus a specific amount of enzymatic activity in the presence of the testswatches. After washing, the swatches are rinsed in runningdemineralized water and air-dried. The reflectance of the test swatchesis measured with a photometer.

The following example is provided to further illustrate the presentinvention and is not intended to limit the scope of the invention in anyway.

EXAMPLE 1 Identification of an Unknown Isolate

Strain ML207a is a Gram-positive, alkaliphilic bacterium isolated fromMono Lake, a hypersaline, alkaline lake situated in California, U.S.A.(Javor, B., in Hypersaline Environments; Springer-Verlag, Berlin andHeidelberg, (1989), pp. 303-305) by plating out (on Medium A, AppendixA) mud and water samples collected in May, 1990. Strain ML207a is acoccus, forming bright yellow-orange, circular, entire, convex colonieson alkaline nutrient agar (Medium A).

Strain ML207a was examined using 22 of the minimum tests listed in Table14. The character states were determined and identification scoresobtained using the MATIDEN program. The results are outlined in Table19. These indicate a very satisfactory identification of strain ML207ato Cluster 1, despite assigning only 22 of the 32 character states fromthe minimum discriminatory tests.

A Willcox probability of 0.9997 was calculated, which is significantlyhigher than the limit set at 0.95. Willcox probabilities of 0.85 and0.95 have been accepted as criteria for a successful identification,(Williams, S. T., et al., (1983), supra; Priest, F. G. and Austin, B.,(1988), supra). A taxonomic distance from the cluster centroid of 0.423is acceptable and within the cluster radius defined at 0.539 (99%level). The standard error of the taxonomic distance at 2.076 is lessthan the upper value of +3.0 suggested by Sneath, P. H. A. ((1979), pp.195-213). In addition, the coccus-shaped cells and yellow-orange colonycolor of strain ML207a also conform with the characteristics of Cluster1 (Table 13).

TABLE 19 Example of the Output from the MATIDEN Program to Identify anUnknown strain against the Identification Matrix Reference number ofunknown is ML207a. Value in Un- Percent in: Character known Best TaxonNext Best Taxon  [10] Gelatin n.t. 99  1  [14] Fumarate − 20  1  [15]Fructose − 60  1  [19] Galactose + 20  1  [24] N-acetylglucosamine −  125  [27] D-saccharose −  1  1  [28] Maltose − 20 25  [32] Acetate − 2075  [36] D-glucose − 20 25  [37] Salicin −  1 25  [38] D-melibiose −  1 1  [42] Propionate −  1 75  [44] Valerate − 20 50  [48] Glycogen −  1 1  [50] L-serine + 40  1  [63] Chymotrypsin + 40 25  [70] β-glucosidase− 20  1  [74] Serine +  1 75  [77] Arginine n.t.  1 99  [80] Methioninen.t.  1 99  [90] Penicillin G + 99 50  [94] Methicillin + 99 99  [96]Streptomycin + 40 75  [97] Tetracyclin − 80 99 [105] Bacitracin + 99 99[112] N-acetyl-D-glucosamine n.t. 40  1 [116] Cellobiose n.t.  1  1[137] Turanose n.t. 40  1 [139] Methyl pyruvate n.t. 60 99 [140]Mono-methylsuccinate n.t. 40 99 [192] Thymidine n.t. 80 50 [197]Glyceral n.t. 80 25 Isolate ML207a best identification is Cluster 1Scores for coefficients: 1 (Willcox probability), 2 (Taxonomicdistance), 3 (Standard error of taxonomic distance). 1 2 3 CLUSTER 10.9997 0.423  2.076 CLUSTER 2 0.261 × 10⁻³ 0.500 5.55 CLUSTER 3 0.40  ×10⁻⁵ 0.540 6.60 CLUSTER 1 CHARACTERS AGAINST % in Taxon Value in unknown[19] Galactose 20 + [74] Serine  1 + [97] Tetracyclin 80 − ADDITIONALCHARACTERS THAT ASSIST IN SEPARATING CLUSTER 1 from CLUSTER 4 [10]Gelatin 99   1 [77] Arginine 1 99 [80] Methionine 1 99 n.t. = nottested.

EXAMPLE 2 Production of Proteolytic Enzymes

Five alkaliphilic strains (60E.4, 81LN.4, wN10 and wN12) were tested forthe production of proteolytic enzyme(s) in a medium poised at analkaline pH. The experiments were carried out in 2 liter shake flasksprovided with a baffle. Each of the flasks contained 400 ml of Medium S.Medium S had the following composition in g per liter: fresh yeast,8.25; glucose, 1.32; K₂HPO₄, 1.6; CaCl₂, 0.05; MgSO₄.7H₂O, 0.05; FeSO₄,0.005; MnSO₄, 0.0066; NaCl, 40.0. The medium was sterilized at 121° C.for 20 minutes and adjusted to pH 10.5 with sterile 40% Na₂CO₃ solution.The flasks were placed in an orbital incubator rotating at 280revolutions per minute at a constant temperature of 37° C. Samples ofculture medium were removed from the flasks at intervals of 0-5.7 daysfor the determination of enzyme content which is expressed in AlkalineDelft Units (ADU-as described in U.S. Pat. No. 4,002,572).

Table 20 shows the enzyme yield and the pH of the cultivation medium atthe moment at which the measurement of enzyme levels were made.

TABLE 20 Production of Proteolytic Enzymes Strain Strain Strain StrainStrain 60E.4 80LN.4 81LN.4 wN10 wN12 ADU/ ADU/ ADU/ ADU/ ADU/ Day ml pHml pH ml pH ml pH ml pH 0 0 10.5 0 10.5  0 10.5 0 10.5 0 10.5 1 137 9.52 10 16 10 0 10.5 2 10 2 2 9.5 18 10 0 10 3 122 10 3 10 17 10 2 10 4 10110 5 88 10 3 10 15 10 5 10 5 10 7 74 10 14 10 9 58 10.5 5 10 14 10 5 103 10

The results of the test, together with the results shown in Appendix Eclearly indicate the presence of proteolytic enzymes, produced by thealkaliphilic bacteria of the present invention, in the culture broth.

EXAMPLE 3 Wash Performance Test Using Proteolytic Enzymes

Enzyme preparations from the alkaliphilic bacteria were tested in aspecially developed mini-wash test using swatches (2.5×2.5 cm) soiledwith milk, blood and ink (obtained from EMPA, St. Gallen, Switzerland).Two types of fabric were tested; 100% cotton (designated EMPA 116) andpolyester (35%)/cotton (65%) designated EMPA 117). The test swatcheswere submitted to the mini-wash test either with or without apretreatment (“pre-oxidized”). The pretreatment consisted of placing theswatches in a solution containing an anionic surfactant, sodiumperborate and a bleach activator (TAED) and stirring at ambienttemperature for 15 minutes. After this treatment the test swatches wererinsed in running demineralized water for 10 minutes and air-dried. Thistreatment results in the fixation of the remaining soil.

The washing tests were performed in 100 ml Erlenmeyer flasks providedwith a baffle and containing 30 ml of a defined detergent compositionplus 300 ADU (Alkaline Delft Units-as described in U.S. Pat. No.4,002,572) protease to be tested. In each flask were placed two EMPAtest swatches. The flasks were placed in a reciprocal shaking water bath(2 cm stroke) and agitated at 200 revolutions per minute. The test werecarried out at 40° C. for 30 minutes. After washing, the swatches wererinsed in running demineralized water for 10 minutes and air-dried. Thereflectance on both sides of the test swatches was measured at 680 nmwith a Photovolt photometer (Model 577) equipped with a green filter.

The wash performance of the supernatant fraction of cultures of severalalkaliphilic bacteria in European powder detergents was determinedaccording to the method specified above. The supernatant fractions wereconcentrated by ultrafiltration (Millipore CX Agitator or Amicon RA 2000spiral ultrafiltrator) so as to produce an enzyme-containing preparationof at least 300 ADU/ml.

100 ml Erlenmeyer flasks were charged with powder detergent IECdissolved in standard tap water of 15° German Hardness so as to give afinal concentration of 4 g per liter, or IEC (3.2 g per liter) plussodium perborate (0.74 g per liter) and TAED (0.6 g per liter) finalconcentrations in standard tap water.

The composition of the powder detergent IEC was as follows:

Component wt % Linear sodium alkyl benzene sulphonate 6.4 Ethoxylatedtallow alcohol 2.3 Sodium soap 2.8 Sodium tripolyphosphate 35.0  Sodiumsilicate 6.0 Magnesium silicate 1.5 Carboxymethylcellulose 1.0 Sodiumsulphate 16.8  Miscellaneous + water up to 100

Standard tap water is composed of CaCl₂.2H₂O, 0.291 g/l; MgCl.6H₂O,0.140 g/l and NaHCO₃, 0.210 g/l dissolved in demineralized water.

To each flask, two EMPA test swatches were added and sufficientenzyme-containing preparations to give a final activity of 300 ADU. Thefinal volume of the sud was 30 ml. By way of comparison, one flaskcontained no enzyme preparation, which was replaced with sterilebacterial culture medium. The results are shown in Tables 21 and 22.

TABLE 21 Application Washing Trials Performance of ProteolyticEnzyme-Containing Preparation from Alkaliphilic Bacterium 60E.4. AverageRemission of Test Swatches IEC + IEC PERBORATE + TAED EN- % IM- EN- %IM- TEST CON- ZYME PROVE- CON- ZYME PROVE- SWATCH TROL PREP. MENT TROLPREP. MENT EMPA 22.7 34.3 51.0 116 EMPA 12.5 12.9  3.2 12.0 12.7 5.8 116(oxidized) EMPA 20.2 48.1 138.2  117 EMPA 12.8 14.5 13.3 13.0 13.1 1.0117 (oxidized)

TABLE 22 Application Washing Trials Performance of ProteolyticEnzyme-Containing Preparation from Alkaliphilic Bacterium 81LN.4.Average Remission of Test Swatches IEC + IEC PERBORATE + TAED EN- % IM-EN- % IM- TEST CON- ZYME PROVE- CON- ZYME PROVE- SWATCH TROL PREP. MENTTROL PREP. MENT EMPA 20.3 25.9 27.9  116 EMPA 11.4 13.7 19.7 116(oxidized) EMPA 18.8 31.1 65.22 117 EMPA 12.4 13.0  5.3 117 (oxidized)

The results of the trials demonstrate the efficacy of the proteolyticenzymes produced by the strains of the present invention in a detergentformulation and the improved washing performance obtained.

EXAMPLE 4 Production of Starch Degrading Enzymes

Three alkaliphilic strains (60E.4, wN12 and wN16) were tested for theproduction of starch degrading enzymes on a starch containing mediumpoised at an alkaline pH.

The experiments were carried out in boiling tubes (2×20 cm) charged with10 ml of alkaline medium Y. Medium Y had the following composition in gper liter demineralized water: yeast extract (Difco), 1.0; KNO₃, 10.0;KH₂PO₄, 1.0; MgSO₄.7H₂O, 0.2; Na₂CO₃, 10.0; NaCl, 40.0; soluble starch(Merck), 20.0. The tubes were inoculated (5%) with cells grown for 24hours on medium A (see Appendix A) at 37° C. As controls, similar tubesof alkaline medium not containing starch were also inoculated.

The tubes were placed in an orbital shaking incubator rotating at 280revolutions per minute, at a constant temperature of 37° C. for 72hours. The fluid containing the enzyme activity was separated from thecells by centrifugation for 10 minutes at 4000 r.p.m.

The enzyme activity of the supernatant fraction was assayed by measuringthe reducing sugars released as glucose from waxy maize starch andquantified with para-hydroxybenzoic acid hydrazide by using a methodbased on that of Lever, M. (1973), Biochem. Med. 7, 274-281. Thereaction mixture (1.0 ml) contained 0.25% (w/v) waxy maize starchsuspended in 0.1 M sodium carbonate buffer, pH 10 (0.9 ml) andenzyme-containing supernatant (0.1 ml). The assays were carried out at25° C. for 30 minutes and the reaction terminated by the addition of 3ml para-hydroxybenzoic acid hydrazide reagent. After boiling for 5minutes the absorbance at 410 nm was measured in a spectrophotometer.The reducing sugars were measured as glucose equivalents from a standardcurve.

One unit of starch degrading enzyme activity is defined as 1 μg ofreducing sugars measured as glucose released per milliliter per minuteat pH 10 and 25° C.

The number of starch degrading enzyme units formed is shown in Table 23.

TABLE 23 Production of Starch Degrading Enzymes ENZYME units per literMEDIUM Y Strain 60E.4 Strain wN12 Strain wN16 plus starch 33,333 9,0886,158 no starch   404   140   246

The results of the test, together with the results shown in Appendix Eclearly indicate the presence of starch degrading enzymes, produced bythe alkaliphilic bacteria of the present invention.

EXAMPLE 5 Stability of Starch Degrading Enzymes in Detergent

The ability of starch degrading enzymes from strains 60E4 and wN12 towithstand detergents, which is essential for their application intextile desizing, is demonstrated.

100 ml Erlenmeyer flasks provided with a baffle were each charged with30 ml of 0.1 M Na₂CO₃/NaHCO₃ buffer, pH 10.1 containing 0.12 g of sodiumdodecyl sulphate (equivalent to 4 g per liter). To one half of theflasks 0.3 g potato starch (equivalent to 1%) was added.

Each flask was dosed with enzyme-containing supernatant from the teststrain by adding 0.5, 1.0 or 2.0 ml (see Table 24). As a control, thesupernatant fluid was replaced with 1.0 ml water. Immediately afteradding the enzyme, a 0.1 ml sample was removed (time=zero hours) for themeasurement of enzyme activity using the para-hydroxybenzoic acidhydrazide method.

The flasks were incubated with shaking at 25° C. for 2.5 hours at whichtime a second 0.1 ml sample was removed for the measurement of enzymeactivity.

As a comparison the experiment was repeated using a conventionalα-amylase (Maxamyl^(R)) from Bacillus amyloliguifaciens.

Enzyme activity was determined using the reducing sugars methodquantified by para-hydroxybenzoic acid hydrazide described previously.

The results are recorded in Table 24.

TABLE 24 Stability of Starch Degrading Enzymes in Detergent ENZYME-CONTAINING ENZYME SUPERNATANT UNITS RECOVERED STRAIN ADDED (ml)CONDITIONS 0 hours 2.5 hours 0* SDS + STARCH  0  0 60E4 0.5 SDS + <1 321.0 STARCH 13 77 2.0 36 132  66B4 0.5 SDS +  3 33 1.0 STARCH  6 65 2.013 119  Standard §) SDS 27 27 Standard §) SDS +STARCH 29 48 *replacedwith 1 ml water §) 250 TAU Maxamyl ® amylase (one TAU, ThermophileAmylase Unit, is defined as the quantity of enzyme that will convert 1mg starch per minute at pH 6.6 and 30° C. into a product which uponreaction with iodine has an equal absorbance at 620 nm as a solutioncontaining 25 g CoCl₂.6H₂O, 3.84 g K₂Cr₂O₇ and 1 ml HCl in 100 mldistilled water.

The results of this test clearly demonstrate the stability of starchdegrading enzymes, produced by the alkaliphilic enzyme of the presentinvention, in the presence of detergent.

What is claimed is:
 1. A pure bacterial culture useful for production ofalkalitolerant enzymes wherein the bacteria consist of aerobic,Gram-positive, coccoid or short rod-shaped, obligate alkaliphilicbacteria consisting essentially of the following characteristics: a)cells are often found in pairs, exhibiting a characteristic snappingform of cell division so as to give a V-form arrangement; b) no growthbelow pH 8; c) on alkaline-agar, forms opaque, orange colored,punctiform or circular colonies; d) in alkaline-broth, growth (37° C.)is flocculent, with the formation of a sediment and surface ring; e)grows optimally at above 30° C., no growth above 40° C.; f) grows atNaCl concentration of between 0-10%, no growth at a concentration of 12%NaCl; g) grows on yeast extract, peptones and carbohydrates; h) gives apositive response to the catalase reaction test; i) gives a negativeresponse to the following tests: 1) KOH 2) Aminopeptidase 3) Oxidase 4)Gelatin hydrolysis 5) Starch hydrolysis.
 2. A pure bacterial cultureuseful for production of alkalitolerant enzymes wherein the bacteriaconsist of aerobic, Gram-positive, straight or slightly curvedrod-shaped, obligate alkaliphilic bacteria consisting essentially of thefollowing characteristics: a) cells are sometimes found in pairs,exhibiting a characteristic snapping form of cell division so as to givea V-form arrangement; b) no growth below pH 8; c) on alkaline-agar formsopaque yellow/ochre-colored circular colonies; d) grows between 10° C.And 40° C., no growth at 45° C.; e) grows at NaCl concentration of0-12%, no growth at NaCl concentration of 15%; f) grows on yeastextract; g) gives a positive response to the following tests: 1)Catalase reaction 2) Gelatin hydrolysis; h) gives a negative response tothe following tests: 1) KOH 2) Aminopeptidase 3) Oxidase 4) Starchhydrolysis.
 3. A pure bacterial culture useful for production ofalkalitolerant enzymes wherein the bacteria consist of aerobic,Gram-positive, straight or slightly curved rod-shaped, obligatealkaliphilic bacteria consisting essentially of the followingcharacteristics: a) cells are often found in pairs, sometimes in shortchains of 2 to 4 cells; b) no growth below pH 7.5; c) on alkaline-agar,forms circular, cream-colored colonies; d) grows between 10° C. and 45°C., no growth at 50° C.; e) grows at NaCl concentration of 0-15%; givesa positive response to the following tests: 1) Catalase reaction 2)Oxidase reaction 3) Gelatin hydrolysis 4) Starch hydrolysis; d) gives anegative response to the following tests: 1) KOH 2) Aminopeptidase.
 4. Apure bacterial culture useful for production of alkalitolerant enzymeswherein the bacteria consist of aerobic, Gram-positive, obligatealkaliphilic bacterium consisting essentially of the followingcharacteristics: a) cells are initially spherical, developing into shortrods, exhibiting a characteristic snapping form of cell division so asto give a V-form arrangement; b) no growth below pH 8; c) onalkaline-agar, forms circular, convex, entire, opaque, orange to deepsalmon pink colonies; d) grows at 10° C. to 37° C., no growth at 40° C.;e) grows on yeast extract; f) grows at NaCl concentration of 0-12%; g)gives a positive response to the following tests: 1) Catalase reaction2) Aminopeptidase 3) Gelatin hydrolysis; h) gives a negative response tothe following tests: 1) KOH 2) Oxidase reaction 3) Starch hydrolysis.